Turns out I don't need to transfer sbb42 into a new vector, since it's already in AK. Also, I won't need transfered sbb09 (3' TR for Tn5) until step two of assembly, so I'll go ahead and Eco/Bam transfer all the other parts I need until I can get that PCR to work.

Map of 1968 Eco/Bam transfer

DNA payload assembly: Eco/Bam transfer

Tim's Phusion MM seemed to work better than mine. I'll do a small fragment zymo and digest the PCR product I made with the Phusion MM (on the left).
This will need to be digested, along with the following...

To Eco/Bam digest

Location

Bjh2271

Amy\'s part Box

sbb06

Romeo H3

sbb12

Romeo F4

jtk2642

jtkplasmid12 A2

iGEM10_005

Conor\'s box (using clone #1)

iGEM10_010

Conor\'s box (using clone #1)

iGEM10_017

Perfect parts box

NOTE: sbb06, sbb12 and the sbb09 PCR product are going to require small Zymo clean-ups!!!

Started digest at 8:00pm.

Gel purified all digests but 1 and 2. I ran the gel for 12 minutes, but that was probably too long for my ~250bp parts. I could barely see the band at 476bp for #4. I'll have to redo the digest tomorrow. Digests are stored in Amy #2.

I still have my digests from my 2345+43/44 assembly, so I'll start from the ligation step. And even though I have sequenced off of tetR promoter yet, I'll also redo the assembly of 42 (tetR-self lysis T) w/ 43/44 while I'm at it. And I'll transform Bjh2017 (SelfLysisT) which is currently in R 1601KC into jtk159 to get rid of the methylation so I can Eco/Bam transfer it into a new vector, in case 42 is messed up.

The following ligations were started at 7:50pm using previously digested products:

Redo of SOEing rxn with A+B

I must have cut out the wrongs bands in my SOEing products, since my Eco/Bam mapping of my minipreps yielded ~3000bp bands... very odd. I remember think the band I was cutting out (from the Zymo product) may have been too low, but the reaction probably worked since the analytical gel I ran before zymoing the products looked liked this:

I don't know what happened between my Zymo and the gel purfication of that Zymo, but I remember the top band being lower than the band in the above picture. Either way, I'm redoing the reaction and cutting out the band at around 6-7K.

Ran an analytical gel of the SOEing PCR product and saw multiple bands, so I ran all my Zymo'd product and gel purified the upper bands. Started Eco/Bam digest of three PCR products and Tahoura's 1968 MP at 2:00pm.

Ligated my PCR products with Pmll-AC and Tahoura's with Pmll-KA. Ligation started at 3:35pm. Plate at 4:50pm.

To do tomorrow

Do Tahoura's Eco/Bam transfer at the same time. The mp's are in my perfect parts box.

Make more phusion MM for Josh. (dntps in enzyme 2 box in -80)

Re-transform 45,46,47,48 mps

Sequencing results

iGEM254

igem10_045 fwd off tetR

igemten063

Fairly short read, but the 700bp that read well perfectly aligned with the region of SL after TetR

iGEM255

igem10_045 rev off pcon

igemten064

good read, but bad alignment. only about 100bp aligned. very garbled.

iGEM256

igem10_046 fwd off tetR

igemten063

Great. 900bp of perfect alignment after tetR.

iGEM257

igem10_046 rev off pcon

igemten064

Pretty crappy read. Can\'t infer much from bad alignment.

Conclusion: igem10_045 probably has large deletions, and I wouldn't be surprised in igem10_046 does too, but I want to resubmit it for sequencing to confirm.

Analytical gel of SOEing A and B PCR products

Lane

Transposase

SOEing Rxn

Expected length

1

Tn5

A

1452

2

SB

A

1454

3

PB

A

1449

4

Tn5

B

5046

5

SB

B

5002

6

PB

B

5860

They all look great!

Second step of SOEing

I'm going to Zymo all of the PCR products, elute with 33uL (PCR was 36uL, I used 3uL for the analytical gel), and then use 1uL of A and B for template in the next PCR along with 1uL of ca998 and g00101.

SOEing Histags with linkers in front of Transposases

The previous oligos I designed to do this by quikchange didn't include linker scar sequences after pelB or before the transposase. This could have hindered proper folding of the protein due to decreased flexibility.

igemTen065

gccggcgatggccGGATCTcatcatcatcatcatcatggatctATAACTTCTGCTCTTCA

60

fwd Quick change his6tag into iGEM10_020 WITH SCAR

igemTen066

TGAAGAGCAGAAGTTATagatccatgatgatgatgatgatgAGATCCggccatcgccggc

60

rev Quick change his6tag into iGEM10_020 WITH SCAR

igemTen067

cggcgatggccGGATCTcatcatcatcatcatcatggatctGGTAAATCTAAAGAAATCT

60

fwd Quick change his6tag into iGEM10_013 WITH SCAR

igemTen068

AGATTTCTTTAGATTTACCagatccatgatgatgatgatgatgAGATCCggccatcgccg

60

rev Quick change his6tag into iGEM10_013 WITH SCAR

igemTen069

gccggcgatggccGGATCTcatcatcatcatcatcatgGATCTGGTTGCTCTCTGGA

57

fwd Quick change his6tag into iGEM10_131 WITH SCAR

igemTen070

TCCAGAGAGCAACCAGATCcatgatgatgatgatgatgAGATCCggccatcgccggc

57

rev Quick change his6tag into iGEM10_131 WITH SCAR

Note: igemten067 may not work... just notice that the mp of the right homologous region is only 46*. Don't know how that happened... maybe try 45 too?

Ah... there's a scar seq in btwn pelB and the transposase which will be included in the annealing region... This bumps up the annealing Tm a bit.. I think I'll run everything at 50 and 55deg.

Note: Phusion is more efficient, but expand is better for genomic DNA.

SOEing Reaction A

Transposase

Goal Part

Template

Oligos

PCR program

Expected length

Tn5

iGEM10_152

igem10_020

ca998/igemTen066

2K55

1452

SleepingBeauty

iGEM10_153

igem10_013

ca998/igemTen068

2K55

1454

PiggyBac

iGEM10_154

igem10_131

ca998/igemTen070

2K55

1449

SOEing Reaction B

Transposase

Goal Part

Template

Oligos

PCR program

Expected length

Tn5

iGEM10_152

igem10_020

igemTen065/g00101

6K55

5046

SleepingBeauty

iGEM10_153

igem10_013

igemTen067/g00101

6K55

5002

PiggyBac

iGEM10_154

igem10_131

igemTen069/g00101

6K55

5860

Checking Self-Lysis

From meeting notes email:

use the following oligos to sequence off 45,46,47,48 to check to see
if self-lysis is intact
igemTen063 GATGCTGTAGGCATAGGCTTGG 22
igemTen064 GCTAGCAAAGTGCCTAGGAC 20 Rev off selflysis (pCon)
also sequence self-lysis on transposase? (igemten63 would work as
forward..., would have to design another rev oligo)

I'm out of 047 and 048, so I'll have to re-transform if I want more of it.

In the meantime, I'll submit 45 and 46 for sequencing w/ igemten063 and 064.

Since bands are of fairly equal brightness, I used 5uL of b8 and 1uL of a6 elutions. Put reaction in thermocycler at 50oC for 1hr.

Transformed jtk030 cells and plated on Amp.

His-tagged Transposase

Colony check:

jc012 vector insertion: several colonies grew on igem10_055 and sbb04. The plates were Amp, though, so there's a bit of fuzzy background. I'll try to pick the center of the large colonies and grow them up in Amp.

Quikchange:

I only got seven colonies on igem10_020 and nothing on 13 or 131. I'll grow up the colonies in AC and miniprep them tomorrow.

What should I do about the ones that didn't work? Options:

Redo it starting with my final PCRs (Dpnl incubation and transformation)

Pick colonies from quikchange plate (grow up in LB-AC), colpcr, no cotransf

Pick colonies from joc12 plate (grow up in LB-A), colpcr, no cotransf (part from PCR)

Update: the strains we were using (jtk030, Righty and Lefty)contain pir under a ptet promoter. So all of our constructs in PMLL (R6K) with parts under tetR were being produced at a low copy number, since the tet repressor was also repressing pir. PLUS they were leaky because the repressor was busy repressing pir in addition to whatever construct (mostly self lysis) that we had under tetR.

Plan of action:

Tim's going to transform 45-52, 20, 13 and 131 into Josh's jtk159 strain, which is pCon-pir. The only thing is that the pir doesn't have a stop codon, but it has proven to still be functional and produce a fairly good copy number.

I'll redo the lysis and transposase assays. I need to plan the assays on saturday, so I can tell Tim how much of what media to grow everything up in for Monday's tests.

End of 1884+1858 project, beginning of histag-GFP-NLS Gibson project

News flash: sequenced the fwd region of igem10_141 (RFP-NLS construct) and found that there's a pelB in front of the part. This could be a problem b/c RFP may not fold in the periplasm. I'm going to add IPTG to a liquid culture and see if it turns red.

His tag will get cleaved off along w/ pelB, so we need to design an oligo to remove pelB from the front of RFP.

Designed the following oligos to set up a Gibson reaction that would ultimately make {<ffGFP>}{<NLS!} in pjc012.

Mapping/Sequencing of 1884+1858 in jc012

Designed oligos for quikchange to add in His6tag. I'm going to wait to ligate my pcr product digest with mike's plasmid digest so I can do it in parallel with my quikchange transformation. Those colonies probably won't be stable for very long since the transposase will be expressed in the cytoplasm.

End of SL/VB Assembly

Sequencing results allowed me to add the following parts to my perfect part box: igem10_045a, igem10_049a, igem10_050c. I'm going to run a Tecan assay to compare lysis of the T-lysozyme version to the L-lysozyme version.

Today, I'll transform jtk030 cells with the minipreps so I can run a Tecan assay comparing the T4 and Lamba lysozyme versions of self-lysis as well as do a macro test to see if they all work in TB, LB, sea water.

Started rescue at 7:45pm.

Redo Map of 1884+1858

Eco/Bam digest of 1884+1858 combo minipreps (e and f a-b)
Started at 2:206pm

Eco/Bam digest resulted in one band. Forgot that I destroyed the Bam site with ligation of Bam/Bgl sticky ends.

Transposition Buffer: Added 1.0165mg to each mL of NEB2 (MgCl2 tetrahydrate is 203.3g/mol and we're attempting to copy a transposition buffer that contains 150mM of MgCl2). We added 36.6mg of MgCl2 to 32.4mL of water and 3.6mL of 10xNEB2 to make 150mM MgCl2 NEB2. We also added 5 drops of diluted HCl (tube labeled diluted HCl... we made it my dipping a pasteur pipette in the 12M HCL and then in 50mL of mgH20) to bring the pH from 7.8 to 7.6 or so.

Spun down all 5mLs of cells and 1uL of control cells (no ara). Resuspended in NEB2+MgCl2 buffer. Transferred control into a tube. Combined all 5mL resuspended cells into one tube, and then separated into 5 tubes.

Added 1uL of atc and 10uL of arabinose to each mL of cells at 1:05pm. Put in 37deg shaker.

Started adding DNA at 1:40pm. For DNA, we mixed two minipreps of 9145-1144. Well, we mixed them after adding DNA from one of the MPs to the 1uL tubes and the 13 .5uL tube (Whoops).

Somehow, we ran out of MP, so we're only adding up to 10uL of DNA.

Time points:
.5hr- 2:10pm
1hr- 2:40pm
1.5hr- 3:10pm
2hr- 3:40pm

Transform into TG1 cells. We mixed 11 tubes of TG1 cells together in a Falcon tube.

Self Lysis Test in NEB2+MgCl2

With bacteria grown up from another colony for Transposase Assay (Arabinose added last night around 5:30pm), with took 2mL, spun then down and resuspended in our NEB2+MgCl2 buffer. I divided the 2mL into 2 tubes and added 10uL of arabinose (just to keep it similar to the above experiment) and 1uL of atc to one of them.

We'll let it sit in the shaker for 1 hr, and then we'll spin down the cells, zymo the supernatant, transform jtk030 (pir+) cells and them plate on Gen.
1:24pm.

Transposase Purification

(1)http://www.springerlink.com/content/u0011pj34254227h/fulltext.pdf
"Transposase: The transposase is a hyperactive triple-mutant version of the Tn5
transposase. The mutations are at residues 54 (E to K), 56 (M to A) and 372 (L to
P) (1). The enzyme can be purchased from Epicentre Technologies (see Note 1).
N-terminal His-tagged and maltose-binding protein-fusion versions of the
hyperactive transposase have also been constructed and used successfully (Yigit,
H. and Reznikoff, W. S., unpublished) (18)."

For a typical reaction, 2 μl (approximately 0.2 μg of protein/μl) of Tnp was added to 18 μl of pRZTL1 plasmid (approximately 1 μg of DNA) in the transposition reaction buffer (0.1 M potassium glutamate, 25 mM Tris acetate, pH 7.5, 10 mM Mg2+acetate, 50 μg/ml bovine serum albumin, 0.5 mMβ-mercaptoethanol, 2 mM spermidine, 100 μg/ml tRNA; final concentrations). The reaction was incubated for 1 h at 20 °C and then diluted 2–3-fold in the same buffer and transferred to 37 °C. This procedure was performed to facilitate binding in the presence of CHAPS present in the reaction mixture as a component of the Tnp storage buffer (TEGX, 10 mM CHAPS). Dilution increased the cleavage reaction presumably due to dilution of CHAPS. CHAPS can be eliminated from the reaction (and storage buffer), and a simple incubation at 37 °C with no dilution is satisfactory if Qiagen-purified DNA is used. We nevertheless used the above two-step procedure for all experiments to synchronize the start of cleavage.

I picked four colonies of each of the 41/42 and 43/44 combinations (a good number of colonies grew up on all of the plates) and four colonies of the restreaked 1884+1858 in jc012 vector. I checked them all for cotransformation, but skipped the colPCR since the parts are so large and jco12 doesn't have ca998/goo101.

Conor and I also picked colonies in order to set up for the Transposase Assay tomorrow. We're going to perform the assay in the following buffer's:

Results from Tn5 Toxicity Assay

If Tn5 is actually being expressed over the course of these 6 hours, then these results would suggest that it's not toxic, since the OD isn't dropping, suggesting death and lysis, but is gradually increasing as the bacteria continue to grow slowly. One of the samples has the same curve but is shifted down, which is probably a result of inaccurate pipetting, which lead to a lower starting OD. As expected, the OD of my controls LB and LB+ara, are low and constant.

1884+1858 in jc012 restreaking

Since these plates made lawns with only a few tiny green colonies, I went back and swabbed the area of the plate with a green colonies and dipped it in 100uL of 2YT and then plated all the 2YT on Amp. Hopefully this will give me larger, isolated green colonies.

Tecan Assay: Testing Self-Lysis timing of 047/048 in LB and Toxicity of 020

Assembly of 1888+1858 with Mike's plasmid

Messed up yesterday's assembly, and realized I don't need to gel purify the digest of Mike's plasmid, because I'll be able to select for the correct plasmids, since the colonies will be green So I set up the following:

41/42+43/44 plates Analysis and colony picking

I'll pick 8 colonies off 42+44 (I have a feeling a lot of them are co-transformed), and 4 colonies off 41+43. Inoculate in LB-AK, colPCR?, streak on triple antibiotics.

Ran a 7KcolPCR

Restock 9145-1144

Transformed jtk030 cells.

1884+1858 ca998/iGEM048

Expected length: ~700bp. They all look great!

I'll also digest Mike's plasmid (Mel001 p3co12) with Bam/XhoI. Started at 2:14pm.

Even though I haven't gotten sequencing results back, I'm going to digest sample c and d with Bgl/XhoI. Digest started at 3:06pm. Gel purified. Expected lengths: 700bp dropout, vector should be ~5700bp. (Single cut would produce ~6400bp) Hopefully the gp worked well... I cut out the upper band, which was too high to really distinguish the length.

Ligation started at 5:29pm.

Transformed jtk030 cells.

Found my digests in the incubator. Turns out I used the undigested eluted PCR product for the assembly. Whoops. Trashed the cells. I'll redo the digestion+ligation tomorrow.

Transposase Assay: Testing Transposase Expression time and Activity Time

Day before, add one colony to 6mL of LB-CK.

Day of assay, move 1000uL of cells into five flasks (can discard extra mL)

Sequencing Results

iGEM10_042a is perfect. (ca998 sequencing redo produced a perfect partial)

Degradation Assay

Conor will do this tomorrow:

Add 1uL of atc to each 1mL sample of cells. Let cells lyse for an hour. Add 1uL of DNA. At each of the following intervals: 15min, 30min, 45min, 1 hour, 1.5, 2, 2.5, 3, remove 200uL of DNA, spin down, and zymo supernatant. After you have all your zymo'd samples, plate on Amp.

Picked two colonies of jtk030 cells transformed with iGEM10_047 and 048 and put them in TB-KA in the shaker upstairs. Tomorrow, I'll see how long it takes for the cells to undergo self-lysis. Each part has the exact same SL device under the same Ptet promoter, so they shouldn't vary.

PCR off 1884+1858 in 1601CK

I designed an oligo that will add a XhoI site to the NLS. So if I do a PCR with ca998 and my oligo igemTen048, hopefully I should get a band around 733bp long. I should only get this band if NLS is after RFP. It can't PCR off just RFP, because there would be no NLS to bind to. And just NLS is only 33bp, so that wouldn't even show up on the gel. Ran a 2K55.

Mapping of 1884 and 1858

I wasn't going to map these, but since sequencing of 1884+1858 a and b w/ g00101 both failed, I'm going to map them. The maps won't show whether or not the NLS tag is there, but it should show how good the MP is and if the backbone is good.

Eco/Bam Digest started at: 12:30pm

Expected lengths: 3142 (vector), 711 (part)

Lanes 1-8 correspond with samples a-h.

Everything looks good. They could have had bad reads because they were cotransformed. I submitted 1884+1858c and d for sequencing.

Sequencing Results

iGEM10_020b in pmllCA is perfect
iGEM10_042a in pmllAC's fwd read was messed up. I submitted the exact same part w/ ca998 again.
iGEM10_041b in pmllCA is perfect

I have left over righty digest of iGEM10_043/044, so I only have to set up a Lefty digest of iGEM10_042/041. NOTE: iGEM10_042 hasn't been sequence confirmed yet, but I'm going to go ahead with assembly and keep my fingers crossed.

I think I started it around 2:00pm.

Started ligation at 4:37pm. Used 2114+2275/2316 digests from construction of 47 and 48.

Oligos

ColPCR results

Expected length: about 900bp. They all look good. Mapping won't show me much, since the NLS is so small (~30bp). So I'm going to sumbit two samples for sequencing, using the g00101 oligo. If they're cotransformed, I'll get back a mixed read.

Colony picking: 42/41PCR+pMLL4AC and iGEM10_020

Picked 8 colonies of iGEM10_020 and checked for cotransformation. Didn't run a colPCR b/c the part is too big.
Picked 4 colonies of 42/41PCR+pMLL4AC and started a 4K colPCR. Had to make more colPCR MM.

Colony Picks

Assembly: iGEM10_020 and Eco/Bam 41 and 42 PCR products into pMLL4AC

Digests:

eco/bam: 41, 42

R: bgl/xhoI of igem019-gel purify (mapping at same time): 6181, 1607. Cut out top band.

L:bam/xhoI of igem001

digests started at 12:46pm

Gel purified 19b,d: There was a band at around 6000bp, but I couldn't see one at 1607, but that could be because I ran the gel for a long time. I gel purified the top band anyway and I'll continue on with the next step of assembly. I could map again, but I think my analytical colPCR combine with sequencing results will be a pretty good confirmation.

Zymo'd 41, 42, and 1.

Ligations:

41/42+pMLL4AC, transform into lefty
19b/d with 1, transform into jtk030

See if i have any ef1a MPs that mapped well that I could submit for sequencing. I want one w/o the double T deletion. However, it's prob before the atg used as a start codon, so it may not affect anything. We'll see...

On a second glance, mapping data for ig114/2271+2017 could be ok. Maybe do with iGEM10_043/044 (four reactions total) and submit them for sequencing?

Nuclear Localization check

pjc012 backbone

Bgl/XhoI transfer of CDS of our protein

his6tag
prom-rbs-his6-my gene!

rfp+nls: 1884+1858 in 1601KC

Transformed into jtk049. Started rescue at 3:35pm. Plated on KC.

Tomorrow, pick colonies. Can do colPCR off plasmid (1601 series).

Next day: miniprep, map, sequence? PCR on bgl/xhoI sites.

may need to pcr a new bam/xhoI around GFP-Nls and then cut into mike's plasmid

Even though my ef1a/atub+2-93 colPCR results don't look so hot, I'll miniprep the ones that weren't co-transformed and map them tomorrow.

ColPCR results for ig114/2271+2017 picks

This analytical backbone PCR seems useless, since a 900bp band was supposed to reveal the presence of the p15 origin of rep, a sign that the part was in the hybrid vector but my negative control, a part in pMLL4AC, also produced a 900bp band! My only choice of analysis then is mapping, but all of the colony PCRs look very crazy. Maybe I'll just miniprep and map them all anyway. Another route I could do would be to perform an Eco/Bam transfer into a vector w/ diff antibiotics, and then do another Eco/Bam transfer into the PMLL4AC. That's just going take a long time...

th395/th396

Lane

Part

Don\'t want

1--4

2271+2017

~900bp band

4--8

ig114+2017

9

2345 In pMLL4AC

ca998/g00101

'

'

'

Lane

Part

Expected length

1--4

2271+2017

2895

4--8

ig114+2017

3126

Minipreped everything and started an analytical Eco/XhoI digest at 5:21pm.

iGEM name

Digest

Expected length

Expected length if still in hybrid

iGEM10_042

Eco/XhoI

3768, 1672

4702, 1672

iGEM10_041

Eco/XhoI

3998, 1672

4932, 1672

One of these is clearly still in its hybrid vector, but I don't know what's going on with the single bands. I'm going to Fusion PCR off ___ using ca998 and g00101. Then I'll Eco/Bam that into PMLL4AC.

One Pot Assembly of atub/ef1a with jh2093

NOTE: I'm intentionally creating a hybrid vector! I think this will save time. Just make sure not to transform into Righty, because then I won't be able to Eco/Bam the product into a good vector. Also, this ef1a has a two base pair deletion, so it may not be functional.

None of the colonies picked yesterday from the iGEM10_047, 48, 51, 52, or 16+2294 were co-transformed. And since the ColPCR didn't reveal much of anything, I'm just going to miniprep the first two samples of each, except for iGEM10_047. iGEM10_047's d sample looked great on ColPCR, so I'll miniprep sample a and d instead of a and b. Also, somehow all 12 of the colonies that I picked for the transfer of 2271/ig114+ 2017 from the hybrid into PMLL4AC seemed to still be in the hybrid vector, based on my analytical pcr (they all had bands around 900bp). I'll miniprep four of each anyway and map them. If they're not the right side, I'll do the transfer over again.

To do:

Minipreps

Ask Tim about Ef1a sequence.

Picking colonies w/ Payload and Payload delivery

30µL into ea mL TB media (use TB for growing up for assay)

Miniprep Map

Tube

Mapping

iGEM name

Digest

Expected length

Expected length if still in hybrid

Good?

1,2

2271+2017 w/ PMLL4AC

iGEM10_042

Eco/XhoI

3768, 1672

4702, 1672

Possibly. Upper band looks good. Perhaps lower band too faint

3,4

ig114+2017 g.p. a w/ PMLL4AC

iGEM10_041

Eco/XhoI

3998, 1672

4932, 1672

Possibly. Upper band looks good. Perhaps lower band too faint

5,6

2345 in PMLL4 b w/ 2114+2316 g.p. a

iGEM10_047

Eco/Bam

5533, 2877

a,b

7,8

2345 in PMLL4 b w/ 2114+2275 a (7/14)

iGEM10_048

Eco/Bam

6102, 2877

a,b*

9,10

1968 in PMLL4 b w/ 2114+2316 g.p. a

iGEM10_051

Eco/Bam

5763, 2877

a,b

11,12

1968 in PMLL4 b w/ 2114+2275 a (7/14)

iGEM10_052

Eco/Bam

6332, 2877

a.b

13,14

16a w/ 2294

Eco/Bam

3166, 2745

righty methylated!!

good for one cut, but need to do Eco/XhoI

Submitted a sample a for each of the 2345/1968 assemblies for sequencing, except I submitted sample d for iGEM10_047.

16+2294 is righty methylated. That's why I only got one band. It's the right sized band though. I'll map it with Eco/XhoI. Started digest at 4:10pm.

Lane 1: 16+SLa
Lane 2: 16+SLb

Expected lengths: 4644, 1466

Analysis: I think the top band is parent vector. Don't know why sample B has three bands... very strange. I submitted a for sequencing.

Everything grew up very well. The only concern I have is that the colonies with 2345 in the part have a transparent loop around the colony, possibly suggesting that self-lysis is starting. I'll have to careful when I miniprep these.

I pick colonies, ran a normal colony pcr for all of them, ran an analytical pcr to make sure the parts I transfered out of the hybrid vector aren't still in the hybrid vector, inoculated and check for co-transformation for the assembly plates. Here are my notes:

pick four colonies off each plate.

run colPCRs w/ ca998/g00101 for all of them

run colPCRs w/ th395/th396 on ig114+2017 and 2271+2017 vector transfers and original minipreps to check for hybrid

Timing wise:

make colPCR MM for 5 rxns w/ th395/396.

set up tubes for colPCR (need 28 tubes for regular, and 8+2=10 for th395/396 version)

don't forget to add template DNA from original minipreps for th395/6 PCR for comparison check.

Analytical PCR results for Transfer from hybrid vector

This is a gel from a colPCR with oligos th395/th396 of four colonies picked from 2271+2017 and ig114 7/16/10 plates. If the plasmid is still a hybrid, it will contain the p15 origin of replication, and will PCR off a ~900bp DNA. This gel suggests that all the colonies I picked contained the hybrid vector. Tim said that if I need to do the transfer over again, I should use more pMLL vector (~3uL). For now, I'll pick more colonies.

Tube

Part

1a-4a

iGEM10_042

5a-8a

iGEM10_041

9a

2271+2017 in hybrid vector

10a

ig114+2017 g.p. in hybrid vector

ColPCR

This results aren't very good. But I think that's to be expected when parts are about 6k or greater. I'm going to mostly ignore these results and miniprep a couple samples from each reaction anyway.

The analytical PCR makes it seem like all the hybrid transfers are still in the hybrid vector... :(

-Eco/Bam digest ig114+2017 g.p. and 2271+2017 g.p. Run on gel and gel purify. Ligate to pMLLAC pre-digested vector.
(calculated expected lengths before running gel, to make sure it's worth the trouble. Bands might be so similar that maybe just a zymo is better)

-ligate g.p. 2294 with g.p. 16a.

-assemble SL-L with vacuole buster:

Bjh2345 in pMLL4-AC with iGEM10_043 and iGEM10_044 (I'm going to use my 2114+2275 miniprep from 7/13, even though it hasn't been sequenced yet. It mapped well, but this doesn't say much since Pcon is so small. Even so, I'm do the reaction anyway and trash it if the sequence comes back bad, like the last one did.)

Bjh1968 in pMLL4-AC with iGEM10_043 and iGEM10_044

NOTE: can't use one pot because iGEM10_043 and iGEM10_044 are NOT methylated. I transformed jtk030 cells to make them. I'll have to do separate righty and lefty digests.

Sequence Analysis of Ef1a/Atub

iGEM57-ef1a

ef1a animal promoter (iGEM10_021 in PMLLKA) miniprepped 7/14

ca998

Deletion of two T\'s at around 410bp. Would cause a frame shift, so it\'s useless.

iGEM58

atub animal promoter (jh2342 in pMLLKA) miniprepped 7/14

ca998

Perfect partial. The read was very short. Only the first 200bp or so were good.

So where to go from here?

I'll submit atub for sequencing with g00101. As for ef1a, since my ef1a in the AK vector (was supposed to be KA... argh) sequenced perfectly. I'll just Eco/Bam Transfer it? Then I'll get colonies tomorrow, and I can ColPCR/inoculate and then miniprep on Tuesday. OR I could pick more colonies today. However, even if I map well, the part could have mutations...

When I pick colonies, I'll run two ColPCRs: ca998/g00101 and th395/th396. If the plasmid is still a hybrid, it will contain the p15 origin of replication, and will PCR off a ~900bp DNA. I'll also run the ColPCR on colonies that I know contain the hybrid so that I can compare. This will allow me to do less minipreps. I'll still map them though, to confirm the vector size.

Important Mini-meeting update: Issue with SL Assembly probably resulted from use of 2017 in 1601 vector

Mapping Yesterday's Minipreps

Ran out the gel a bit more, and I think it shows that the two 2271+2017 samples mapped well:

Lane

Miniprep

Digest

Expected Lengths

'

Cut out

Good?

1

2114+2275a

Eco/XhoI

4627, 1466

good

2

2114+2275b

Eco/XhoI

4627, 1466

good

3

2271+2017a

Eco/XhoI

3768, 1672

too big

4

2271+2017b

Eco/XhoI

3768, 1672

too big

5

ig114+2017e

Eco/XhoI

3998, 1672

too big

6

ig114+2017f

Eco/XhoI

3998, 1672

too big

7

ef1a in PMLLKA b

Eco/Bam

2868, 897

good

8

atub in PMLLKA b

Eco/XhoI

2405, 1598

good

9

atub in PMLLKA c

Eco/XhoI

2405, 1599

bad

10

jh2294 in PMLLKA

BglII/XhoI

3777, 1607

Upper band

good

Sequencing Results for the rest of the Minipreps from my first batch of colony picks from Gel-Purified Assembly

Submission Name

Part

Oligo

Analysis

iGEM51

2114+2275 g.p. F

ca998

BAD- a bunch of junk. No alignment with sequence.

iGEM52

2114+2275 g.p. R

g00101

Perfect partial. The last 1000bp or so of the part is perfect

iGEM53

2271+2017 g.p. F

ca998

Perfect Partial- first ~1000bps of the part is right

iGEM54

2271+2017 g.p. R

g00101

Perfect Partial- last ~700bp of part look good

iGEM55

ig114+2017 g.p. F

ca998

About 1000bp aligned, except for mutation: @ 291bp, mutation: @ 449bp

iGEM56

ig114+2017 g.p. R

g00101

Perfect Partial: last 700bp sequenced perfectly

The boundaries of parts 2114+2275 and ig114+2017 look good. I'm going to PCR off the middle to check if the insides are go too. I used ca56 for ig114+2017 (binds to pbad promoter). I designed a primer for the middle of

I picked colonies, inoculated and ran a colony pcr for all the parts we moved into the new, CORRECT KA vector.

Important notes:

5 and 11 were originally in AK vectors. 10 was also in an AK vector, but I gel purified it. Even so, I will run an analytical PCR of my minipreps of 10, and I ran an analytical Colony PCR with th313 and g00101 for my 5 and 11 colonies. I picked 12 colonies of 5 and 11.

I also check for parent vector bleed-through by plating all the parts on spec, except for 9 (plated on triple antibiotics since it came from CK) and efla+atub (PCR products)

All the inoculations are in the shaker.

The colony PCRs are in the black PCR machines. They'll probably be done around 6pm.

IMPORTANT UPDATE: TUBE MARKED PMLL6 (KA) in Eco/Bam Box is INCORRECT

Christoph just ran and analytical digest/gel that proved that the vector in the tube that was supposed to contain KA actually contained AK vector instead of KA. I need to Eco/Bam transfer the parts into a new KA vector. Also, this explains why the assembly with the Self-Lysis Device didn't work. We need to Eco/Bam transfer it into a KA vector too.

Digesting the new pMLL-KA backbone

Marianna gave us 10uL of her CMED7 in KA part. The dropout will be 324bp. To digest all of it, I multiplied the Analytical Mapping protocol by 2.5:

Ligation of ef1a and atub (bjh2342) eco/bam zymo'd digest

Ligated ef1a and atub eco/bam zymo'd digests (from my previous insertion into the supposed KA vector) with the new gel purified Eco/Bam digested KA vector. Started at 7:05pm. Transformed jtk030, MC1061 pir+ cells. In retrospect, I could have used Lefty.

Checking vector backbones of SL/VB Eco/Bam transfers

Set up a 2K55 PCR reaction with Taq MM, 1uL of 1/10 th313 oligo (in the middle of AmpR) and iGEM10 (rev oligo right before EcoR1 site) to verify the order on antibiotics on the backbone. This reaction PCR's off the AmpR gene to right before the EcoRI site. So if the vector is actually A-, the PCR product will contain a bit of AmpR and plasmid backbone and will be 865bp. If the plasmid is -A, the PCR product will contain a bit of AmpR, lots of plasmid backbone and the other antibiotic. In the case of CA, this amounts to 1808bp.

See Eco/Bam page in Excel notebook.

This data suggest that all the backbones that should be A- are indeed A- and that all the backbones that are -A are indeed -A. So the antibiotics shouldn't be the problem. For whatever reason, nothing turned up in the 2345 lane. I'll run the PCR again just to double check.

Colony PCR of SL/VB and 16a+SL Assembly

Results:

Very odd. Even though I gel purified the correct bands, it appears that only 2114+2316 worked correctly. I'm going to check to make sure that everything is in the correct vector, because I don't know what else could be causing a problem. Since I'm miniprepping a the couple correct products, might as well miniprep and map a few others to try and figure out what the problem may be.

Cotransformation check

Can disregard the results for 16a+SL since all samples were cotransformed. 2017 combo inoculations are useless since nothing showed up on the colony pcr. Very strange, since they weren't co-transformed. GOOD NEWS: atub and ef1a look great.

Redo of SL/VB and 16a+SL digests with Gel Purification

To avoid co-tranformation and hopefully encourage the creation of the parts I want, I'm going to run a preparative gel, select the appropriate bands and gel purify.

Performed a Lefty (Bam/XhoI digest), Righty (Bgl/XhoI digest), and one-pot digest on all the parts in my SL/VB promoter as well as iGEM10_016a and Bjh2294 in PMLL-KA can be found in the Cheshire Chat plate in H3 and F3. I'll gel purify the Lefty digests of Bjh2345, Bjh2271, Bjh1968, Bjh2114, ig114, 16a and the Righty digests of Bjh2316, Bjh2275, and the two self-lysis devices. The other digests are just to make sure everything was methylated correctly and that the one-pot reaction works on these vectors.

Notes on protocols:
Used Manual 2ab Assembly protocols for Lefty and Righty Mastermixes. Used my One-pot recipe, except with 0.4uL of each enzyme. Also, the DNA taken from the Cheshire Cat Plate was diluted by half b/c it was eluted with 100uL, so we had to adjust the amounts of NEB2 and enzyme used so that the recipe in the R/L digests turned out to be 1.6uL NEB2 and 1.5uL of each enzyme total, since our using 8uL of DNA instead of 4uL of DNA would otherwise have diluted the buffer and enzyme.

Digests started at 3:35pm.

From left to right: Lefty Digests, Righty Digests

Triple Digests

Results:

Lane

Part

vector

Purify

L Fragments (if no methylation for R\'s)

R Fragments (if no methylation, for L\'s)

One Pot fragments (if not methylated)

Proper methylation?

1

ig114a

pMLL7-AK

L-Upper

2925, 1196

2440, 1681

1681, 1244, 1196

Yes

2

Bjh2114

pMLL9-CA

L-Upper

1518, 1270

1476, 1312

1476, 1270, 42

Yes

3

Bjh2271

pMLL7-AK

L-Upper

2695, 1196

2210, 1681

1681, 1196, 3891

Yes

4

iGEM10_016a

pMLL5-CK

L-Upper

2333, 1196

2054, 1475

N/A (not methylated)

5

Bjh2316a

pMLL7-AK

R-Upper

4491, 1196

4006, 1681

2810, 1681, 1196

Yes

6

Bjh2275a

pMLL7-AK

R-Upper

5060, 1196

4575, 1681

3379, 1681, 1196

Yes

7

Bjh2294 F3

pMLL9-CA

R-Upper

4114, 1270

3777, 1607

2507, 1607, 1270

Yes

8

Bjh2294 H3

pMLL9-CA

R-Upper

4114, 1271

3777, 1608

2507, 1607, 1271

Yes

NOTE: lane 2 in the lefty gel is Bjh2294 H3, and everything else is pushed over one lane

Summary: Everything is methylated as it should be. I cut out all the right bands and purified them. Also, this shows that triple digestion isn't very efficient and cause a lot of parent vector bleedthrough (the top band of all the lanes). Also, note how dilute the SL DNA is... I'll still elute with the standard 8.5uL to concentrate the DNA, but this is still a potential problem.

Ligation and Transformation of SL/VB and 16a+SL purified digests

Started ligation at 6:45pm. Will transform into jtk030 (pir +, MC1061 cells). I forgot to digest 2017, so I used a righty digest that I had done previously. Started rescue at 8:10pm.

All the samples from 16a,b + SL were cotransformed, and since Jin mentioned that making a mastermix for the onepot reaction may result in there not being enough enzyme per reaction, I'm going to do it over again without making a mastermix AND using 0.5uL of enzyme instead of o.3uL of enzyme. This should cut back on parent vector bleedthrough as well.

There are a significant number of colonies on the plate for the 2271+2017 and ig114+2017 redo. I'll pick colonies/colony pcr/inoculate/check for cotransformation for 8 colonies from each. Also, since all the colony PCR's failed for my re-picking of 2114+2275, I'm going to redo the one-pot reaction for that as well.

The ligation of the atub promoter to the KA vector (Bjh2342-pMLL6-KA) was very successful, since I got a lot of colonies. I'll inoculate and run colony PCR (no need to check for co-transformation. Since my two colony picks of ef1a seemed to fail on the colony PCR yesterday, I'm going to pick a bunch more, inoculate, and run colony PCR again.

SL/VB Assembly: Redo of One Pot (with more enzyme) for 2114 combos

One Pot (used 0.4uL of enzyme):

2114+2275

2114+2316

Will be ready for ligase at 2:00pm. Added ligase at 2:00pm. Will be ready for transformation into RIGHTY cells at 2:30pm. Rescue started at 3:30pm.

Put in incubator at 12:14pm. Ready for Zymo at 1:14pm. Zymo'd and added ligase at 2:00pm. Will be ready for transformation into RIGHTY cells at 2:30pm. Rescue started at 3:30pm.

Possibly successful assemblies (Animal Promoter, SL/VB and iGEM20

Picked colonies (8 each)/inoculate/cotransformation check

2271+2017

ig114+2017

16a+SL (pick at least 12 NEW colonies)

atub and ef1a (no cotransformation check)

Miniprep of Payload Delivery Vectors

Miniprepped Payload Delivery stuff (in shaker w/ Mg LB)

Sequencing Results for iGEM10_017b, 2114+2316b,Bjh2345 PMLL4

Sequencing results:

iGEM10_017b: the first 990bp or so of the 1546bp part are perfect, so I think it's safe to say that this miniprep is good to use for future assembly.

2114+2316b: this proved to have the Pcon, but no vacuole buster in the part. At first I thought this was odd, since the part miniprepped well, but it makes sense now. The part had a bad colony PCR since only the 43bp pCon was in the vector. The part had what looked like a good miniprep map since the vector was the same size as the part, and I when I saw a band, I assumed it was the part AND the vector. My mistake.

Bjh2345 PMLL4: bad read. Do again, but make sure concentration is accurate?

Animal Promoter: Colony PCR of Ef1a

Conor Eco/Bam digested and zymo'd the atub promoter (Bjh2842). Started ligation with PMLL6-KA vector at 4:45pm. Transformed into jtk049 cells. Started rescue at 5:55pm.

Payload Delivery

Since the payload delivery device contains the Self Lysis device under a promoter that's induced in low levels of Mg, I need to grow up my colonies in LB containing 30mM Mg to prevent Lysis. In the future, I should also rescue/plate on Mg. I selected clear colonies.

Tomorrow, I will miniprep the inoculations and map the minipreps next to the parent vector containing the part, so I can make sure the whole part is in the new vector. (Jin said recombination events can occur which could shorten the part)

SL/VB Promoter Assembly

Redo one pot reaction with 2271+2071 and ig114+2071

Conor set up two one pot reactions and put them in the thermocycler under my ONEPOT program. Added ligase at 4:45pm. Will transform into LEFTY cells. Started rescue at 5:55pm.

Started ligation at 4:05pm. Ligated unmethylated 6+24 digested with BglII+XhoI with both unmethylated iGEM10_016a+b, which had been digested with BamHI+XhoI. Tubes are labelled 16a+SL and 16b+SL (SL meaning Self Lysis, which is part 6).

After ligation is over, I will transform into RIGHTY cells. Started rescue at 5:05pm. Plated on CA.

After four hours have passed since you first added arabinose, add 1uL of atc per mL of cells. Allow 1 hour for lysis.

Will add 0.9uL of atc, since 900uL of cells are being used and the concentration of the atc is 1000x.

After that one hour, add 5uL of DNA to each tube.

Remove a 200uL sample of lysate after 30min, 1 hour, 1.5hour, 2hours.

Immediately after removal, spin down the lysate and zymo the supernatant.

Transform lefty pir+ cells with the lysate. Plate on Amp and Gen.

Unfortunate update: Sequencing proved that iGEM10_020b is a dud. Gibson didn't work because one of our junctions contained palidromic terminal repeats, so the oligos designed based off those junctions didn't have a unique binding site. Oh well. We'll proceed with manual 2ab assembly.

iGEM10_20 2ab Assembly

Mapping of iGEM10_016 minipreps a and b

An Eco/Bam digest of iGEM10_16 in PMLL5-CK should result in bands at 2662 and 867.

Note: iGEM10_017 has already been mapping. (see below) Results were slightly confusing, so I'll submit it for sequencing...? iGEM10_001 was also mapped, and looks good. 1a-c are all good options.

Started digestion at 4:55pm.

Both have bands at the appropriate places, but 16b has a lot more parent vector remaining, for whatever reason, so 16a is probably the best choice.

Assembly of iGEM10_018

Lefty Digest of iGEM10_16

Righty Digest of Self Lysis Device (6+24 taken from well G10 of ROMEO plate)
Started at 4:42pm. Zymo'd and stored in my working box.

SL/VB Promoter Manual Assembly: Analysis of One-Pot reaction

2114+2316 and 2114+2275 both successfully transformed righty pir+ cells. The plates are covered in colonies. 2271+2017 and ig114+2071 weren't as successful. The former had no colonies, while the latter had 11, which is surprising, because I used barely any DNA.

I will pick four colonies from each, colony pcr, inoculate, and check for cotransformation.

Cotransformation results:

The ones with letters in the corners were chosen for minipreps.

Tube

Combo

Expected length

Good?

co-transformed?

miniprep?

1

2114+2316

3053

yes

no

yes

2

\"

no

yes

3

\"

4

\"

no

5

ig114+2017

3125

yes

no

yes

6

\"

yes

7

\"

yes

8

\"

9

2345 in pMLL4

2880

no

yes

10

\"

no

yes

11

\"

no

yes

12

\"

no

13

2114+2275

3622

14

\"

no

yes

15

\"

no

yes

16

\"

Moving into jh1601K

Performed an Eco/Bam Digest of jh1601K. Put in incubator at 11:09am. Trashed that digest. Realized I need to do a gel purification, so I'll do the digest again with 4uL of plasmid.

Started digest again at 4:54pm.

2345 Redo from 24+PMLL4 digests

The plate is covered in colonies. I will pick 8 colonies, inoculate, and check for cotransformation.

Eco/Bam transfer 2348 and 2291 into 1601K

2345 Redo: Ligating Tim's ebid 24 and EB pMLL4-AC

Since my Colony PCR turned out so horribly yesterday, and 2345 is the Self Lysis Device we've been using for all our construction, I'm just going to use Tim's digests and ligate them together.

Started ligation at 11:48am.

I transformed the plasmid into lefty pir+ cells. Started rescue around 12:30pm. Plated on AC at 2:13pm.

One Pot Assembly of Jin's Parts

I'm going to redo the construction of the following with a one pot reaction, since they're all methylated.

Lefty Digest

Righty Digest

Transform into

Plate on

Short Description

'

2114

2316

R

CK

pCon.VacuoleBusterA

2114

2275

R

CK

pCon.VacuoleBusterB

2271

2017

L

AC

Pbad.SelfLysisT

ig114

2017

L

AC

Ptet.SelfLysisT

Started digest at 11:10am, for all but ig114+2017. I added the speck of 2017 (~.2uL) left at around 11:36am. Started the 20 min heat kill for all but ig114+2017 at 12:30pm. Accidently set the heat kill program for 20 sec instead of minutes and added ligase. Hopefully that won't hurt anything. Put all four tubes in the PCR machine to heat kill at 12:53pm. Added ligase at 1:41pm. Started rescue at 2:42pm.

These numbers have been based on Qiagen minipreps of Jin's pBjh1601
assembly vectors: